High precision isotope ratio mass spectrometry (IRMS) measurements can be made on three fundamental levels: bulk stable isotope analysis (BSIA), molecular or compound-specific isotope analysis (CSIA), and intramolecular or position-specific isotope analysis (PSIA). Despite its widespread use in all areas of natural science, there are no instruments for high throughput molecular or intramolecular analysis of natural isotope variability. We have recently overcome major technical barriers by demonstrating on a proof-of-principle basis high precision comprehensive 2-dimensional-GC-based GCxGCC-IRMS for ?13C and by microfabricating highly robust fused silica reactors compatible with temperatures greater than 1500C. Here, we seek to exploit these breakthroughs by expanding GCxGCC-IRMS beyond ?13C to enable high throughput ?15N, ?18O and ?2H analyses and by developing a system for high precision intramolecular isotope analysis; the resulting instrumentation will be applied to investigate hypothesis-driven, high throughput isotopic metabolomics of fatty acids and intermediary metabolites, lactate and acetate, with natural isotope and/or artificial tracer techniques. The Specific Aims are (1) Design and construction of a GC-pyrolysisxGCC-IRMS for high precision intramolecular analysis by adaptation of a cryogenically modulated GCxGCC-IRMS. (2) Implementation of interfaces for molecular (CSIA) and intramolecular analysis of 2H/1H, 15N/14N, and 18O/16O; Evaluation of fragment isotopic fidelity after pyrolysis for 2H/1H PSIA for non-exchangeable H. (3) Applications of PSIA and CSIA to fatty acids and related molecules: Natural molecular and intramolecular isotope ratios of n-3 polyunsaturated fatty acids (PUFA) in existing samples from humans and experimental systems to test hypotheses regarding dietary origin of n-3 and n-6 long chain PUFA, possibly establishing a novel biomarker for 22:6n-3 biosynthesis, and showing how biotransformation of any dietary component can be measured with natural intramolecular isotope ratios. Tracers. Use of 13C and ?2H in labeled acetate, lactate, and PUFA to understand the kinetics of 13C flux among fatty acids; lactic acid ?13C and ?D in existing human samples will also be used to test the hypothesis that this molecule exhibits intramolecular isotopic structure. Relevance to human health: Natural isotope variability at the molecular and intramolecular levels is a vast, nearly untapped repository of biochemical information within organisms. Molecular and intramolecular isotopes, the subject of isotopic metabolomics, are natural biomarkers of molecular precursors, and of physiological state that hold information complementary to metabolomic/metabonomic information, for instance, about molecular flux. Because natural isotopic variability requires no prior dosing or infusions, convenient applications to clinical testing are likely. Finally, the expansion of rapid, highly sensitive intramolecular analysis capabilities to stable isotope labeling studies expands the use of a routine method in biomedical research. PUBLIC HEALTH RELEVANCE: Enabling technology for high throughput and robust determination of high precision intramolecular isotope ratios, also known as position-specific isotope analysis (PSIA), in biomolecules is proposed for C, N, H, and O. The research plan capitalizes on adaptation of comprehensive two dimensional gas chromatography instrumentation as well as innovative components to create a robust system. Applications to polyunsaturated fatty acid and lactic acid metabolism are pro- posed.